RID 101: Basic Principles of Radial Immuno-Diffusion Everything you wanted to know about RID testing *But were afraid to ask*

Transcription

1 RID 101: Basic Principles of Radial Immuno-Diffusion Everything you wanted to know about RID testing *But were afraid to ask* Carl A. Ascoli, Ph.D., CSO 1 2/29/2016

2 RID 101: Basic Principles Immunologicals Group 2 What Is RID?? RID is an acronym for a laboratory-based testing procedure which stands for: Radial Immuno-Diffusion.

3 What is RID Used For? 3 RID is typically used to determine the quantity, or concentration, of a specific antigen (i.e. protein) in a sample usually serum or plasma* using antibodies. *Sample could be any biofluid : serum, plasma, milk, saliva or purified protein solution.

4 How Does RID Work? 4 An antigen (either the specimen, calibrator, or control) is applied to a cylindrical well cut within an agar gel plate containing a specific antibody. The plate is then incubated at 22 C for a specified period of time (usually between 48 and 120 hours). The antigen diffuses radially through the agarose gel containing a mono-specific antibody and an antibody/antigen complex develops, as a precipitin ring.

5 How Does RID Work? 5 The precipitant ring that forms can be physically measured and is translated into a quantitative result. Physical measurement can be made digitally or by eye.. Digital RID Plate Reader Jeweller s Eyepiece using a micrometer

6 Data Analysis 6 Calculating Results: Linear relationship exists between the ring diameter squared and concentration of antigen. By constructing a calibration curve (i.e. a graph) using dilutions of various calibration materials of known concentrations, unknown concentrations can be read off and calculated. Results are then used for measuring/determining the protein concentrations contained in the test material.

7 Who Uses RID? 7 Basic research laboratories in academia, private, government and biotech/ biopharma laboratories Cell culture laboratories growing monoclonal antibodies Laboratories wanting to quantify analytes before purification. Clinical and Forensic Laboratories performing analytical measurements Researchers needing to measure BSA in complex solutions. Manufacturing facilities performing QA/QC checks on processes.

8 What Competes with RID? 8 Analyte ELISA kits do quantify analytes like RID, but the process is more costly, more complicated and requires more complicated analysis. Other companies market RID kits, but these kits are not as user friendly or optimized for cell culture production. Other types of kits that measure analytes may be subject to interference caused by other analytes present in suspensions. Other types of kits to measure analytes require specialized equipment to read or process the samples.

9 Why Use RID? 9 Easy to use. Can measure an analyte without purification. All encompassing, self-contained kit pretty much everything required is included. No special equipment/instrumentation is needed. Sensitive and specific test results. Over 2,000 peer reviewed citations confirm performance.

10 What is in a RID Kit? 10 RID Plates (14 well/tests per plate). Calibration Material (lyophilized). DI Water (for reconstituting Calibration Material). Sample Diluent (for use with Calibration Material & test samples). Package Insert / Instructions.

11 Why are Calibrators Needed? 11 Calibrators are used as a quantitative reference calibrators have a known protein concentration which will produce a specific precipitant ring diameter at this given concentration. Calibrators are also used to create a Calibration Curve by testing various calibrator dilutions on a RID plate (i.e. 100%, 75%, 50%, and 25%), a linear graph can be various dilutions. This graph is then used to calculate concentrations of unknown samples.

12 Why Use the Provided Diluent 12 & DI Water? Both eliminate potential test errors due to water quality. #1 problem in test performance as recorded by Technical Services are those traced back to customers using poor water. Ensures accuracy of test results. Required component as stated within the Instructions For Use.

13 Production of a RID plate 13 Using reference materials known concentrations, tests are conducted to find the optimum volume/plate of antibody to give a specific ring diameter. Liquid agar is mixed with measured antisera and poured onto the base RID plate via calibrated pump. Plates are poured in a clean room to minimize risk of contamination. When gel is cooled and set, wells are punched into plate and QC test plates are checked for the correct ring diameter using reference calibrator.

14 Final QC Testing 14 All finished components are tested together. Calibrator should give correct ring diameter on plate (+/- 0.2mm). Controls within +/- 10% of assigned value. External standard/panel samples tested if available.

15 What Products are Available? 15 A line of research-based RID kits focused on the detection animal proteins, namely Bovine Albumin & IgG and Mouse Immunoglobulins (IgG & IgM) and the IgG sub-classes (IgG1 IgG2 & IgG3).

16 Products 16 PRODUCT DESCRIPTION TESTS per KIT CATALOG NUMBER Bovine Albumin NL-RID Kit 42 RN204.3 Bovine IgG LL-RID Kit 42 RL200.3 Bovine IgG NL-RID Kit 42 RN200.3 Mouse IgG1 NL-RID Kit 42 RN273.3 Mouse IgG2a NL-RID Kit 42 RN274.3 Mouse IgG2b NL-RID Kit 42 RN275.3 Mouse IgG3 NL-RID Kit 42 RN276.3 Mouse IgM NL-RID Kit 42 RN278.3

17 Related Products 17 PRODUCT DESCRIPTION CATALOG NUMBER Mouse TrueBlot Western Blot Kit Anti-Mouse IgG γ1,2a,2b,3 Peroxidase Conjugated Bovine Serum Albumin, 30% Solution C46 BSA-30 Blotto Immunoanalytical Grade B Anti-Bovine IgG (H&L) Fluorescein Conjugated Protein A Purification Assay Development CUST22M CUST35

18 For More Information 18 Our helpful Technical Service staff is ready 24/7 to provide assistance. More technical information can be found on our website at